Volatile Constituents from Different Parts of Three Lamiacea Herbs from Iran.

The essential oils obtained by hydrodistillation from the stem, leaf and flower of Phlomis aucheri Boiss., which is endemic to Iran, stem, leaf and root of Teucrium polium L. and solvent free microwave extraction oil from leaf of Ajuga chamaecistus Ging. Subsp chamaecistus were analyzed by GC and GC/MS. Germacrene D (11.10%, 28.31% and 21. 06%) was the main constituent in the stem, leaf, and flower oils of P. aucheri, respectively. The other main component in the stem oil of the plant was (E) - anethole (24.58%) and in the flower oil was β- caryophyllene (15.93%). All three oils were rich in regard to sesquiterpenes. The main components in the stem, leaf and root of T. polium were α- muurolol (25.02%, 20.03% and 19.53%), α- cadinol (15.72%, 8.11% and 13.01%) and β-cayophyllene (10.86%, 10.11% and 10.64%) respectively. All three oils were rich in regard to sesquiterpenes. The major components in the leaf oil of A.chamaecistus were (z)-β-ocimene (12.11%) and germacrene D (10.11%). The oil of the plant was rich in regard to both monoterpens and sesquiter penes.


Introduction
The genus Phlomis is comprised of about 100 species, 17 of them are described in the flora of Iran, among which 10 are endemic (1, 2).
Teucrium polium is a perennial shrub, 20-50 cm high, distributed widely in the dry and stony hills and deserts of almost all Mediterranean countries, South western Asia, Europe and North Africa.
In the traditional Iranian medicine, T. polium tea is used to treat oilments such as abdominal pain, indigestion, common colds, and urogenital diseases (16).
The genus Ajuga is represented in the flora of Iran by five species in which Ajuga chamaecistus has contained several endemic subspecies including A. chamaecistus ssp. chamaecistus (1, 2).
Several biological studies have been performed on many species of this genus which have confirmed their ethno pharmacological properties such as hypoglycemic (29), anti inflammatory (30), anabolic, analgestic, antiarthritis, antipyretic,hepatoprotective, antibacterial, antifungal, antioxidant, cardiotonic (31), and antimalarial (32) properties and also their application in the treatment of join diseases (33) .
Our study deals with the analysis of the oils from stems, leaves, and flowers of Phlomis aucheri, from stems, leaves, and roots from Teucrium polium and also from leaves of Ajuga chamaecistus growing wild in Iran.

Plant materials
The stems, leaves, and flowers of Phlomis aucheri, which is endemic to Iran, and stems, leaves and roots of Teucrium polium, were collected from Salehabad area, Province of Ilam, west of Iran, both in July 2013, during the flowering stage. The leaves of Ajuga chamaecistus were collected from Mehran, Province of Ilam, in July 2013. Voucher specimens have been deposited at the Herbarium of the Reasearch Instituents of Forests and Rangelands (TARI), Tehran, Iran.

Solvent-free microwave extraction
Solvent-free microwave extraction (SFME) of the leaf of A. chamaecistus was performed in a Milestone ETHOM 1600 batch reactor, which is a multimode microwave reactor operating at 2455 MHz with a maximum delivered power of 1000 W, variable in 10 W increments. The dimensions of the PTFE coated cavity are 35×35×35 cm. During the experiment, time temperature, pressure, and power were controlled using the «easy-WAVE» Software package. Temperature was monitored with the aid of a shielded thermocouple (ATC-300) inserted directly in to the sample container.
In a typical SFME procedure, 250 g of dry leaves of A. chamaecistus were moistened prior to extraction by soaking in water for 1 h, then draining off the excess water.
This step is essential to give the leaves the initial moisture. Moistened leaves were next placed in the reactor without any added solvent or water. The essential oil is collected, dried with anhydrous sodium sulphate and stored at 0 °C until used.

Gas chromatography analysis
Gas chromatography analysis was performed on Schimadzu 15A gas chromatograph equipped with a split /splitless injector (25 °C) and a flame Note: a Compounds listed in order of elution from HP-5 MS column; b Retention indices to C 8 -C 24 n-alkanes on HP-5 MS column. ionization detector (250 °C). Nitrogen was used as carrier gas () and the capillary column used was DB-5 (50 m × 0.2 mm, film thickness 0.32 . The column temperature was kept at 60 °C for 3 min and then heated to 220 °C with a 5 °C rate and kept constant at 220 °C for 5 min. Relative percentage amounts was calculated from peak area using a Schimadzu C-R4 A chromatopac without the use of correction factors.

Gas chromatography -mass spectrometry analysis
Analysis was done using a Hewlett-Packard 5973 with a HP-5 MS column (30 m × 0.25mm, film thickness 0.25). The column temperature was kept at 60 °C for 3 min and programmed to 220 °C at a rate of 5 °C and kept constant at 220 °C for 5 min. The flow rate of Helium as carrier gas with () MS was taken at 70 e V.
The retention indices for all the components were determined according to the Van Den Dool method, using n-alkanes as standard (41,42).
The compounds were identified by (RI, DB5) with those reported in the literature and by comparison of their mass spectra with the Wiley library or with the published mass spectra (43).

Results and Discussion
The composition of the essential oils from stems, leaves, and flowers of Phlomis aucheri, stems, leaves and roots of Teucrium polium and leaves of Ajuga chamaecistus, are listed in Table  1, 2 and 3, respectively, in which the percentage and relative retention indices of components are given.
(E)-Anethole (24.58%) was the other main component in the stem oil of the plant and not detected in the leaf and flower oils. As can be seen from the above information, all three oils were rich in regard to sesquiterpenes (51.52%, 78.52% and 85.16%), respectively. The monoterpene fraction was relatively small, representing only 12.32%, 8.88% and 1.70%, respectively. In the stem oil of P. aucheri, considerable percentage of non terpenoid compounds, compairing to other parts of the plant oils, were identified (27.74%). In an earlier study, Javidnia et al. analyzed the essential oil of the aerial parts of P. aucheri collected in Fars province. The oil was found to be rich in caryophllyene oxide (33.5%), β-caryophyllene (27.0%) andβ-selinene (9.9%) (44).
Only a few reports on the analysis of essential oils of Phlomis species have been published.
Water distilled essential oils from aerial parts of P. persica and P. olivieri, which are endemic to Iran, have been the subject of our previous studies. The major components of both oils were germacrene D (38.2% and 26.4%) and bicyclogermacrene (16.3% and 12.7%), respectively.
Camparison of the present results with those of our previous investigation of oils of the Phlomis genus showed that they are also dominated by sesquiterpenes.
As it is shown from Table 2, 45 components representing 96.84%, 41 constituents representing 98.48% and 20 compounds representing 88.7% were identified in the oils of stem, leaf and root of T. polium, respectively.
The monoterpene fraction of the stem and leaf oils was relatively small, representing (6.73% and 21.45%) of the total oils, resepectively. In the root oil of the plant we could not find any trace of monoterpenes. In the root oil of T.polium, considerable percentage of non terpenoid compounds, compared to other parts of the plant oils, were identified (28.74%).
Water distilled oil obtained from the aerial parts of T. persicum, which is endemic to Iran, have been the subject of our previous studies. epi-α-Cadinol (23.2%) and α-pinene (17.3%) were the main components among the thirty-one constituents characterized in the oil of T. persicum representing 95.9% of the total components detected (55). The oil of T. gnaphalodes was characterized by higher amounts or β-caryophyllene (12.1%), sabinene (8.8%) and trans-pinocarveol (7.8%) (53).
The qualitative and quantitative variation between our results and our previous reports (40) for the constituents of the oil from aerial parts may be attributed to the different environment conditions and different methods of extraction of the oils.
The oils of the genus Ajuga have been the subject of only a few studies. The oil of A. chamaecistus subsp. tomentella contained thymol (34.5%) and exo-fenchol (15.6%) as the major components (56).

Acknowledgment
We are grateful to Dr.V. Mozaffarian Note: a Compounds listed in order of elution from HP-5 MS colum; b Retention indices to C 8 -C 24 n-alkanes on HP-5 MS column.